1. Field of the Invention
The present invention relates to sequences useful in the control of gene expression and, in particular, to sequences for promoting keratinocyte-specific gene expression.
2. Description of the Related Art
Keratinocytes are cells found exclusively in stratified squamous epithelia, including epidermis, tongue, esophagus, cervix and cornea (for review, see Watt, 1988). As surface and lining cells, keratinocytes have several unusual structural features in common, including (a) 10 nm keratin intermediate filaments (IFs), which constitute a major part of the keratinocyte cytoskeleton, (b) desmosomes, which are membranous plaques responsible for interconnecting keratinocytes as a cellular sheet, and (c) a group of inner membrane envelope proteins, which become covalently cross-linked by transglutaminase when keratinocytes undergo terminal differentiation. In addition, differentiating keratinocytes are the natural cell type-specific hosts for a family of circular, double-stranded DNA viruses known as papillomaviruses, although expression of HPV genes has also been detected in relatively undifferentiated cervical carcinomas in vivo as well as simple epithelial-like cervical carcinoma cells in vitro (for a review, see Broker and Botchan, 1986).
Despite minor differences, mitotically active keratinocytes from different stratified squamous epithelial tissues appear to be quite similar not only in morphology but also in biochemistry (Nelson and Sun, 1983). The major proteins expressed by these cells are a pair of keratins, K5 (58 kd) and K14 (5 kd), which form the 10 nm cytoskeletal filaments (Nelson and Sun, 1983; Eichner et al., 1986). In differentiating keratinocytes, the major proteins are also keratins, but in this case, the keratins are expressed in a tissue-specific fashion (for reviews, see Moll et al., 1982; Sun et al., 1984). For epidermis, terminally differentiating cells in vivo express keratins K1 (67 kd) and K10 (56.5 kd) (Fuchs and Green, 1980; Moll et al., 1982; Roop et al., 1983), and suprabasal cells in vitro express K6 (56 kd) and K16 (48 kd) (Kopan and Fuchs, 1989).
Little is known about the molecular mechanisms underlying the expression of genes in basal keratinocytes or the processes controlling changes in keratin and viral gene expression during keratinocyte differentiation. However, a number of recent studies have begun to focus on identifying putative regulatory domains and transcription factors which might be involved in expression of either endogenous or viral genes in keratinocytes (Marchuk et al., 1985; Blessing et al., 1987; 1989; Cripe et al., 1987; Hirochika et al., 1988; Vassar et al., 1989; Chin et al., 1989; Jiang et al., 1990). For papillomaviruses, assessing the relevance to keratinocyte-specific gene expression has been complicated because (a) the viral genomes encode proteins which influence transcription of viral genes and (b) even though HPV genes are preferentially expressed in keratinocytes in vivo, most in vitro studies have been carried out with HeLa or C-33A cells, both of which appear to be relatively undifferentiated, simple epithelial-like cervical carcinoma cells that do not express most keratinocyte-specific structural proteins.
Given this caveat, several HPV genes have been shown to contain sequences (defined to limits as small as .about.30-100 bp) which are not only required for viral expression but also bind epithelial-specific proteins (Cripe et al., 1987; Swift et al., 1987; Gloss et al., 1987; Hirochika et al., 1988; Gius et al., 1988; Chin et al., 1989). In contrast, for genes expressed naturally in keratinocytes, only coarsely defined regulatory domains (300 bp or greater) have thus far been identified (Vassar et al., 1989; Blessing et al., 1989; Jiang et al., 1990).
In the absence of molecular studies, the 5' upstream sequences of a number of genes expressed specifically in keratinocytes have been screened for similarities which might provide clues as to sequences which might be important for gene expression (Marchuk et al., 1985; Tyner et al., 1985; Johnson et al., 1985; Blessing et al., 1987; Lersch et al., 1989). From these comparisons, several putative regulatory sequences have been postulated based upon either extensive similarities to known viral enhancer sequences, e.g. the SV40 core enhancer (Marchuk et al., 1985), the widespread occurrence of a sequence, e.g. the CK 8-mer consensus sequence 5' A A N C C A A A 3' (Blessing et al., 1987; Cripe et al., 1987), or the conservation of a sequence, e.g. 5' T G C A G G T G T G A 3' (SEQ ID NO:2) among two closely related genes (Lersch et al., 1989).
Since so little is known about the sequences and transcription factors involved in keratinocyte-specific expression, the extent to which such comparisons will prove to be valid is almost wholly unknown. Among the questions which remain to be answered are: Is there a common regulatory mechanism controlling expression of a large number of keratinocyte-specific genes? Are papillomavirus and keratinocyte genes regulated in similar fashions? Are the same transcription factors recruited to control gene expression in dividing and differentiating keratinocytes? Clearly, elucidating sequences necessary for keratinocyte-specific gene expression and purification of the proteins which interact with these sequences is a prerequisite to understanding transcription of genes in keratinocytes. To begin to investigate cell type-specific expression in keratinocytes, the functional human genes encoding K14 have been isolated and characterized (Marchuk et al., 1985) and K5 (Lersch et al., 1989). Not only do these genes encode the major keratinocyte proteins, but in embryogenesis, their expression is upregulated coincident with the commitment of an embryonic basal cell to an epidermal fate (Kopan and Fuchs, 1989).
Previously, it has been reported that a human K14 gene construct containing .about.2300 bp of 5' upstream sequence gave specific expression in the basal layers of stratified squamous epithelia in transgenic mice (Vassar et al., 1989). However, this previous study failed to identify the specific regions or nucleotide sequences that might be responsible for conferring specific expression. Therefore, this information does not allow one the ability to multimerize and/or employ smaller sequences and factors that play a role in mediating epithelial-specific expression. These smaller, specific sequences could be used to confer specific expression without resort to the 2300 bp sequence reported by Vassar et al. In addition, multimerization of these sequences might be used to create more powerful expression without loss of tissue specificity. Accordingly, there is a continuing need for the identification of the precise sequences controlling epithelial-specific gene expression. Moreover, there is a need for the identification of the actual stretches of nucleotides responsible for mediating the specificity, as well as a need for the development of control regions having an improved mediating capability.